The code, by which the tertiary structure of a protein is programmed by its amino acid sequence, is of fundamental importance in our understanding of biology at the molecular level. Studies aimed at the reversible folding and unfolding of proteins can give some experimental insight into how this programming works, especially if states intermediate between the native and denatured state are detected. NMR spectroscopy has proved useful in detecting such complex unfolding behavior. This proposal deals with the application of nmr to study the unfolding the refolding of bovine pancreatic ribonuclease (RNase) and derivatives in an attempt to detect such intermediate states. The behavior of many individual amino acid residues will be followed during the unfolding transition. For certain amino acids, this will require chemical modification of the enzyme. NMR unfolding experiments on the proteolytic derivatives of RNase (RNase-S and RNase-P) should shed light on the roles of the N- and C-termini in stabilizing the native structure. All of these experiments should provide information as to the roles played by the various segments of the enzyme in maintaining the native three dimensional structure and also provide information about the pathway by which the enzyme folds into this unique structure. In addition, this proposal seeks to characterize some unusual derivatives of RNase, which were discovered in the course of some preliminary chemical modification experiments required for the nmr work discussed above. These products have 2-4 time the activity of native RNase under standard assay conditions. Although we have not carried out any characterization experiments on the dervatives, our working hypothesis is that they represent RNase-mercaptoethanol mixed disulfides. At the moment, this hypothesis is based mainly on chemical intution. If correct, however, these products would serve as useful models for the study of protein structure at intermediate stages of disulfide bridge formation.